This invention relates to nucleic acid and amino acid sequences of a new human translational regulator and to the use of these sequences in the diagnosis, prevention, and treatment of inflammation and disorders associated with cell proliferation and apoptosis.
Protein synthesis is an indispensable process by which living organisms grow, differentiate, and propagate. The three stages of protein synthesis are initiation, elongation, and termination. In eukaryotes, the first event in protein synthesis is the attachment of a free molecule of methionine (Met) to the end of a tRNA. Met-tRNA and a small ribosomal RNA subunit 40S bind to the mRNA near the AUG initiation codon to form the 40S complex. Addition of the 60S ribosomal RNA subunit to the 40S complex forms the peptidyl-tRNA transfer site. Once the mRNA, 40S, and 60S complexes are in position, peptide synthesis may begin.
Translation initiation factors, eIF1a, eIF2, and eIF3, initiate the formation of the 40S complex and are part of the complex as well. Binding between the 40S ribosomal RNA subunit and the mRNA is aided by eIF4a, eIF4b, and eIF4f using energy from the hydrolysis of GTP bound to eIF2. Translation initiation factor eIF5 promotes the hydrolysis of ribosome-bound GTP producing the energy necessary to bind the 40S and 60S complexes.
eIF4f is a complex which recognizes 5xe2x80x2-mGpppN, the CAP structure of all eukaryotic mRNAs. The complex facilitates the association of the 40S subunit to mRNA for translation initiation. Translation initiation is regulated by the phosphorylation of the CAP-binding protein, eIF4e, a subunit of the eIF4f complex. Addition of insulin to adipocytes or muscle cells increases phosphorylation of eIF4e and stimulates initiation of protein synthesis (Morley, S. J. and Traugh, J. A. (1990) J. Biol. Chem. 265: 10611-10616). Overexpression of eIF4e has been associated with cell transformation (Lararis-Karatzas, A. et al. (1990) Nature 345: 544-547).
PHAS-I, PHAS-II and 4E-BP1 are three regulators of translation initiation (Pause, A. et al. (1994) Nature 371: 762-767; Hu, C. et al. (1994) Proc. Natl. Acad. Sci. 91: 3730-3734; and Lin, T. -A. and Lawrence, J. C. Jr. (1996) J. Biol. Chem 271: 30199-30204). The association of PHAS-I or 4E-BP1 with eIF4e prevents the formation of the active CAP-binding complex, eIF4f. Phosphorylation of PHAS-I or 4E-BP1 by an insulin- or a growth factor-dependent kinase releases eIF4e from a complex with PHAS-I or 4E-BP1 and prepares eIF4e to bind CAP for translation initiation. PHAS-I and PHAS-II are found to have overlapping but different patterns of expression in tissues. Phosphorylation of both PHAS proteins promotes dissociation of PHAS-eIF4e complexes and stimulates cell growth (Lin et al., supra).
The discovery of a new human translational regulator and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of inflammation and disorders associated with cell proliferation and apoptosis.
The invention features a substantially purified polypeptide, human translational activator (TRANAC), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2 or variants thereof.
In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. In another aspect the invention provides a composition comprising an isolated and purified polynucleotide sequence encoding TRANAC.
The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants thereof. In a particular aspect, the polynucleotide sequence is the complement of SEQ ID NO:2. In another aspect the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding TRANAC under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified TRANAC having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist which decreases the activity of a polypeptide of SEQ ID NO:1. In one aspect, the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO:1.
The invention also features a method for treating or preventing inflammation by administering TRANAC, a method for treating or preventing cancer by administering TRANAC, a method for treating or preventing an disorder associated with apoptosis by administering an antagonist of TRANAC, and a method for stimulating cell proliferation by administering an antagonist of TRANAC.
The invention also provides a method for detecting a polynucleotide which encodes TRANAC in a biological sample comprising the steps of: a) hybridizing a polynucleotide sequence complementary to TRANAC (SEQ ID NO:1) to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding TRANAC in the biological sample. In a preferred embodiment, prior to hybridization, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.